Brigham and Women's Hospital Harvard Medical School
Identification of Proteins Functionally Redundant to the Werner's Syndrome Gene Product.
Werner syndrome (WS) is a rare genetic disorder associated with premature aging and increased risks for cancer. The gene underlining WS has been cloned and found to encode a member of the RecQ DNA helicase family. In addition to the drastic mutations found in WS patients, there are also mutations with unknown consequences in the general population. These natural variants (polymorphism) may contribute to the differential rate of aging in the population. Understanding the physiological function of WRN may therefore shed light on not only pathological aging but also natural aging.
We have purified a protein (FFA-1) based on its activity to induce the assembly of DNA replication foci in frog (Xenopus laevis) egg extracts. Sequence analysis indicates that FFA-1 is the homologue of human Werner syndrome gene (WRN), suggesting that WRN may be involved in DNA replication. However, WRN is not an essential gene. We hypothesize that another RecQ helicase family member, Bloom syndrome gene (BLM), may be functionally redundant to WRN.
To test this hypothesis, we have cloned the Xenopus homologue of BLM. We have been able to efficiently express xBLM as fusions to various affinity tags in E. coli. We have demonstrated that the His-xBLM fusion protein possesses DNA helicase activity. The GST-xBLM fusion protein has been used as antigen for antibody production. The antibody has been purified on an affinity column and is currently being used for immunofluorescence staining and immunodepletion studies on xBLM. Preliminary results suggest that xBLM and FFA-1 may indeed participate in the same DNA transaction pathway.